Osteoclasts (OCs) are the only cells capable of bone resorption, a process required for both normal bone homeostasis and pathological bone loss. In the context of tumor metastasis to bone, recruited OCs not only resorb bone, causing pain and fractures, but also change the microenvironment to encourage tumor growth. The resorptive activity of OCs releases bone-stored factors that fuel tumor cells; in turn, tumor cells secrete factors that increase the number and activity of OCs, creating a positive feedback loop for tumor-mediated osteolysis. Understanding the mechanisms of OC activation and how they change the microenvironment to one more favorable for tumor growth is critical for developing treatments for bone metastasis. The NF-?B family of transcription factors is critical for OC differentiation and cell survival. In the first funding period, we determined that the classical NF-?B pathway, mediated by the p65/RelA subunit, is important for OC precursor survival, but not differentiation. In contrast, we found that the alternative NF-?B pathway, mediated by NIK and RelB, controls OC differentiation. Furthermore, absence of either NIK or RelB reduces pathological bone loss in vivo in the context of tumor-mediated osteolysis. Expression of NFATc1 and c-fos, key transcription factors for OC differentiation, is diminished in the absence of RelB, as is the expression of several NFATc1 target genes, suggesting that RelB may modulate osteoclastogenesis via NFATc1 and c- fos. Recently, cIAP and TRAF3 have been implicated as critical negative regulators of NIK, and thus RelB. Drugs blocking cIAPs and disruption of NIK/TRAF3 interaction both result in constitutive activation of the alternative NF-?B pathway. Our central hypothesis is that activation of the alternative NF-?B pathway, via NIK and RelB, controls induction of NFATc1 and c-fos, to enhance osteoclastogenesis and promote a bone microenvironment conducive to osteoporosis and bone metastasis. This proposal explores the effects of constitutive activation of this NIK pathway in the context of bone metastasis, using genetic and pharmacologic approaches, and will address the transcriptional mechanism by which NIK and its downstream effector RelB control osteoclastogenesis. Thus, our specific aims are as follows: Aim 1: Determine the effect of pharmacologic NIK activation by cIAP antagonist treatment on the bone microenvironment at baseline and in the context of bone metastasis. Aim 2: Determine the mechanism by which constitutive genetic activation of NIK, in osteoclasts, affects basal bone homeostasis and tumor- mediated osteolysis. Aim 3: Determine the role of NIK and RelB in the transcriptional control of NFATc1 and c-fos during OC differentiation. Overall, this proposal builds on the findings in the original R01 that RelB, the NF-?B subunit downstream of NIK, is critical for OC differentiation, and extends the studies to understand the transcriptional mechanism for this effect, providing a stronger knowledge base for therapeutic inhibition of the NIK/RelB pathway to prevent bone loss. Because a newly developed class of drugs being developed as anti-cancer agents - the cIAP antagonists -- activates the NIK/RelB pathway, we will also probe the effects of activation of this pathway on bone, focusing on models of bone metastasis. These studies will be critical for understanding and preventing potential adverse effects of these NIK-activating drugs on bone integrity and the incidence/severity of bone metastasis. PUBLIC HEALTH RELEVANCE: Osteoclasts are the cells responsible for removing bone, and their action is necessary for the maintenance of healthy bones. However, they also can cause unhealthy amounts of bone loss is many diseases, including osteoporosis and cancer metastasis to bone. We will study a specific pathway important for the regulation of the osteoclast in normal mice and those with tumors growing in bone.